Throughout this application, various publications are referenced. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
HIV infects primarily helper T lymphocytes and monocytes/macrophages—cells that express surface CD4—leading to a gradual loss of immune function which results in the development of the human acquired immune deficiency syndrome (AIDS). The initial phase of the HIV replicative cycle involves the high affinity interaction between the HIV exterior envelope glycoprotein gp120 and the cellular receptor CD4 (Klatzmann, D. R., et al., Immunodef., Rev. 2, 43-66 (1990)). Following the attachment of HIV to the cell surface, viral and target cell membranes fuse, resulting in the introduction of the viral genome into the cytoplasm. Several lines of evidence demonstrate the requirement of this interaction for viral infectivity. In vitro, the introduction of a functional cDNA encoding CD4 into human cells which do not normally express CD4 is sufficient to render these otherwise resistant cells susceptible to HIV infection (Maddon, P. J., et al., Cell 47, 333-348 (1986)).
Characterization of the interaction between HIV gp120 and CD4 has been facilitated by the isolation of cDNA clones encoding both molecules (Maddon, P. J., et al., Cell 42, 93-104 (1985), Wain-Hobson, S., et al., Cell 40, 9-17 (1985)). CD4 is a nonpolymorphic, lineage-restricted cell surface glycoprotein that is a member of the immunoglobulin gene superfamily. High-level expression of both full-length and truncated, soluble versions of CD4 (sCD4) have been described in stable expression systems. The availability of large quantities of purified sCD4 has permitted a detailed understanding of the structure of this complex glycoprotein. Mature CD4 has a relative molecular weight of 55,000 and consists of an amino-terminal 372 amino acid extracellular domain containing four tandem immunoglobulin-like regions denoted V1-V4 , followed by a 23 amino acid transmembrane domain and a 38 amino acid cytoplasmic segment. Experiments using truncated sCD4 proteins demonstrate that the determinants of high-affinity binding to HIV gp120 lie within the amino-terminal immunoglobulin-like domain V1 (Arthos, J., et al., Cell 57, 469-481 (1989)). Mutational an alysis of V1 has defined a discrete gp120-binding site (residues 38-52 of the mature CD4 protein) that comprises a region structurally homologous to the second complementarity-determining region (CDR2) of immunoglobulins (Arthos, J., et al., Cell 57, 469-481 (1989)).
The HIV-1 envelope gene env encodes an envelope glycoprotein precursor, gp160, which is cleaved by cellular proteases before transport to the plasma membrane to yield gp120 and gp41. The membrane-spanning glycoprotein, gp41, is non-covalently associated with gp120, a purely extracellular glycoprotein. The mature gp120 molecule is heavily glycosylated (approximately 24 N-linked oligosaccharides), contains approximately 480 amino acid residues with 9 intra-chain disulfide bonds (Leonard, C. K., et. al., J. Biol. Chem. 265, 10373-10382 (1990)), and projects from the viral membrane as a dimeric or multimeric molecule (Earl, P. L., et. al. Proc. Natl. Acad. Sci. U.S.A. 87, 648-652 (1990)).
Mutational studies of HIV-1 gp120 have delineated important functional regions of the molecule. The regions of gp120 that interact with gp41 map primarily to the N- and C- termini (Helseth, E., et. al., J. Virol. 65, 2119-2123 (1991)). The predominant strain-specific neutralizing epitope on gp120 is located in the 32-34 amino acid residue third variable loop, herein referred to as the V3 loop, which resides near the center of the gp120 sequence (Bolognesi, D. P. TIBTech 8, 40-45 (1990)). The CD4-binding site maps to discontinuous regions of gp120 that include highly conserved or invariant amino acid residues in the second, third, and fourth conserved domains (the C2, C3 and C4 domains) of gp120 (Olshevsky, U., et al. J. Virol. 64, 5701-5707 (1990)). It has been postulated that a small pocket formed by these conserved residues within gp120 could accommodate the CDR2 loop of CD4, a region defined by mutational analyses as important in interacting with gp120 (Arthos, J., et al., Cell 57, 469-481 (1989)).
Following the binding of HIV-1 gp120 to cell surface CD4, viral and target cell membranes fuse, resulting in the introduction of the viral capsid into the target cell cytoplasm (Maddon, P. J. et al., Cell 54:865 (1988)). Most evidence to date indicates that HIV-1 fusion is pH-independent and occurs at the cell surface. The HIV-1 fusion protein is gp41, the transmembrane component of the envelope glycoprotein. This protein has a hydrophobic fusion peptide at the amino-terminus and mutations in this peptide inhibit fusion (Kowalski, M. et al., Science 237:1351 (1987)). In addition to gp41, recent observations suggest that gp120 plays a role in membrane fusion distinct from its function in attachment. For example, antibodies to the principle neutralizing epitope on gp120, the V3 loop, can block infection without inhibiting attachment (Skinner, M. A. et al., J. Virol. 62:4195 (1988)). in addition, mutations in the tip of this loop reduce or prevent syncytia formation in HeLa-CD4 cells expressing the mutated gp120/gp41 molecules (Freed, E. O. et al., J. Virol. 65:190 (1991)).
Several lines of evidence have implicated molecules in addition to CD4 and gp120/gp41 in HIV-1 induced membrane fusion. For example, recent studies have indicated that human cells may contain an accessory molecule, not present in non-primate cells, which is required for HIV-1 fusion (Dragic, T. et al., J. Virol. 66:4794 (1992)). The nature of this accessory molecule or molecules is unknown. While some studies have postulated it might be a cell surface protease (Hattori, T. et al., Febs. Lett. 248:48 (1989)), this has yet to be confirmed.
Fusion of the HIV-1 virion with the host cell plasma membrane is mimicked in many ways by the fusion of HIV-1 infected cells expressing gp120/gp41 with uninfected cells expressing CD4. Such cell-to-cell fusion results in the formation of multinucleated giant cells or syncytia, a phenomenon observed with many viruses which fuse at the cell surface. Much of our current understanding of HIV-1-induced membrane fusion is derived from studies of syncytium formation. For example, this approach was used to demonstrate that expression of HIV-1 gp120/gp41 in a membrane, in the absence of any other viral protein, is necessary and sufficient to induce fusion with a CD4+ membrane (Lifson, J. D. et al., Nature 323:725 (1986)).
Compared with virion fusion to cells, syncytium formation induced by HIV-1 appears to involve an additional step. First, the gp120/gp41-bearing membrane fuses with the CD4-bearing membrane. This is a rapid and reversible process which connects the membranes at localized sites and allows membrane-bound dyes to flow from one cell to the other (Dimitrov, D. et al., AIDS Res. Human Retroviruses 7:799 (1991)). This step presumably parallels the attachment of a virion to a CD4+ cell and the fusion therebetween. The second stage in cells fusion is the irreversible fusion of cells to form syncytia. The efficiency of this process is increased by the interaction of cellular adhesion molecules such as ICAM-1 and LFA-1, although these molecules are not absolutely required for syncytium formation to proceed (Golding, H. et al., AIDS Res. Human Retroviruses 8:1593 (1992)).
Most of the studies of HIV-1 fusion, including those discussed above, have been performed with strains of HIV-1 which have been extensively propagated in transformed human T cell lines. These strains, known as laboratory-adapted strains, differ in several important characteristics from primary or clinical isolates of the virus obtained from HIV-1 infected individuals (O'Brien, W. A. et al., Nature 348:69 (1990)). Some examples of these differences are listed in the table below.
Laboratory adaptedStrainsPrimary Isolatestropic for transformedmany are tropic forT cell lines, do notprimary monocytes and doinfect primary monocytesnot infect transformedT cell linesvery sensitive torelatively insensitive toneutralization byneutralization by sCD4soluble CD4gp120 spontaneouslylittle spontaneous strippingdissociates from gp41,and sCD4 only causesand this stripping isstripping at 4° C., notincreased by sCD4at 37° C.
These differences are mirrored by differences in the primary sequence of the viral proteins, and in particular of the envelope glycoproteins. In some cases, the different tropisms of primary isolates and laboratory-adapted strains of HIV-1 have been mapped to regions on gp120 such as the V3 loop (O'Brien, W. A. et al., Nature 348:69 (1990)). It is possible that different V3 loops interact with different accessory molecules on T cell lines or monocytes, thereby mediating tropism.
HIV-1 envelope-mediated cell fusion is a model for the early stages of HIV-1 infection and can be used as an assay for anti-viral molecules which block HIV-1 attachment and fusion (Sodroski, J. et al., Nature 322-470 (1986), Lifson, J. D. et al., Nature 323:725 (1986)). Moreover, HIV-1 induced cell fusion is important in its own right as a potential mechanism for the pathogenesis of HIV-1 infections. It is a mode of transmission of HIV-1 from infected to uninfected cells (Gupta, P. et al., J. Virol. 63:2361 (1989), Sato, H. et al., Virology 186:712 (1992)) and by this mechanism, it could contribute to the spread of HIV-1 throughout the body of the infected individual. Cell fusion is also a direct mechanism of HIV-1-induced cell death (Sodroski, J. et al., Nature 322:470 (1986), Lifson, J. D. et al., Nature 323:725 (1986)). Syncytia are seen in vivo, notably in the brains of AIDS patients suffering from neurological complications such as AIDS dementia complex (Pumarola-Sune, T. et al., Ann. Neurol. 21:490 (1987)). In addition, syncytia have been observed in the spleens of HIV-1-infected individuals (Byrnes, R. K. et al., JAMA 250:1313 (1983)). It is possible that cell fusion may play a role in the depletion of CD4+ T lymphocytes that is characteristic of the pathogenic process leading to AIDS (Haseltine, W. A. in AIDS and the new viruses, Dalgleish, A. G. and Weiss, R. A. eds. (1990)).
In this context, it may be significant that HIV-1 isolates from asymptomatic HIV-1-infected individuals often infect cells in vitro without inducing syncytia. In contrast, clinical isolates from patients with ARC and AIDS are commonly highly virulent, syncytia-inducing strains (Tersmette, M. et al., J. Virol. 62:2026 (1988)). In addition, there is often a switch from non-syncytium inducing (NSI) to syncytium-inducing (SI) isolates within patients as the disease progresses and symptoms appear (Tersmette, M. et al., J. Virol. 63:2118 (1989), Cheng-Mayer, C. et al., science 240:80 (1988)). It is not clear why some HIV-1 strains do not induce syncytia, although it is possible that cells infected with these strains do not express sufficient levels of gp120/gp41 for cell fusion to occur, by analogy with some other fusogenic viruses. However, it is believed that this switch from NSI to SI HIV-1 strains influences the clinical course of HIV-1 infection. The presence of naturally occurring anti-syncytia antibodies in some subjects may delay the development of HIV-1 related diseases in these subjects (Brenner, T. J. et al., Lancet 337:1001 (1991)).
The development of methods for measuring HIV-1 envelope glycoprotein-mediated membrane fusion serves a useful role in further elucidating the mechanism of HIV-1 infection, and enabling the identification of agents which alter HIV-1 envelope glycoprotein-mediated cell fusion. At present there exist several potential methods for measuring such fusion.
The first is an assay of HIV-1 envelope glycoprotein-mediated cell fusion in which fusion is measured microscopically by measuring the transfer of fluorescent dyes between cells (Dimitrov, D. S., et al., AIDS Res. Human Retroviruses 7:799-805 (1991)). This technique measures dye distribution rather than fluorescence intensity and as such cannot be performed using fluorometer. The assay would not be easily automated and has not been performed with cells which stably express the HIV-1 envelope glycoprotein.
The second is an assay for HIV-1 envelope-mediated cell fusion measured between (a) cells which stably express the HIV-1 tat gene product in addition to gp120/gp41, and (b) CD4+cells which contain a construct consisting of the β-galactosidase gene under the control of the HIV-1 LTR promotor. When these cells fuse, β-galactosidase is expressed and can be measured using an appropriate soluble or insoluble chromogenic substrate (Dragic, T., et al., Journal of Virology 66:4794 (1992)). This assay takes at least 1 day to perform and cannot easily be adapted to new target cells such as primary macrophage cells. This assay also does not measure cell fusion in real time and is thus not amenable to use in analyzing fusion kinetics.
Finally, the third is a fluorescence dequenching assay for the fusion of HIV-1 virions to cells (Sinangil, F., et al., FEBS Letters 239:88-92 (1988)). This assay requires the use of purified HIV-1 virions, and both the purification of HIV-1 virions and the assay must be performed in a containment facility. It would be difficult to readily isolate sufficient quantities of clinical virus isolates to perform the assay. Furthermore, this assay is more complicated and less reproducible than a RET assay using cells which stably express HIV-1 envelope glycoproteins and CD4.
The methods of the subject invention employ a resonance energy transfer (RET) based assay which overcomes the problems inherent in the above-identified methods for measuring HIV-1 envelope glycoprotein-mediated membrane fusion. Specifically, the methods of the subject invention employ a RET assay which is rapid, reproducible, quantitative, adaptable to various cell types, and relatively safe, and can be automated.